Contraction in all muscles is regulated physiologically by the level of free calcium present. In vertebrate and arthropod muscle, there are regulatory proteins which bind calcium and confer a calcium- dependency to actomyosin interactions with ATP, the rate of hydrolysis being directly related to the concentration of calcium. These regulatory proteins, troponin-tropomyosin, are located on thin filaments. We have found that there is considerable variation in the type of regulatory system used in the animal kingdom. The thin filament-linked system found in arthropods differs from the vertebrate type in molecular weight characteristics and in calcium binding ability. In marked contrast, in the molluscs and other lower invertebrates, the regulatory system is an inherent part of the myosin molecule and no troponin is present. The comparative approach will be continued and extended. Representative organisms previously untested will be analyzed to identify whether their regulatory systems are thin filament or myosin- linked. Native thin filaments and purified myosin will be prepared and characterized by SDS gel electrophoresis, and their respective roles will be assayed by their affect on ATPase activity using a number of interspecies interactions. Specific calcium binding to these components will also be determined. The peptide maps of troponins from various species will also be compared. This work should elucidate the site of calcium regulation in a variety of organisms and lead to a unified hypothesis for the control of actin-myosin interaction. This comparative approach will enable one to determine whether troponin components are conserved or vary during evolution, to accurately describe the evolution of muscle regulatory systems, and to give new information regarding the phylogenetic relationships of animals.